B. Protein Blotting

A general protocol for sample preparation.

Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.

Wash membrane again four times for 5 min each in TBST.

The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

Add antibodies against surface antigens to the assay tubes as per manufacturers’ recommended volume or concentration and incubate for 30 min on ice.

Add 2 ml of Incubation Buffer and wash by centrifugation.

Aspirate supernatant and resuspend in 500 μl of 2% formaldehyde.

Fix for 15 min at room temperature.

Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

Add 1ml of 0.1% Triton™ X-100 (v/v in PBS) to the cell pellet.

Resuspend and let stand for 30 min at room temperature.

Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

Resuspend cell pellets in 100 μl of antibody working solution, diluted according to individual antibody datasheet or product webpage in Incubation Buffer.

Incubate for 1 hr at room temperature.

Wash 2X by centrifugation in Incubation Buffer.

If using a fluorochrome-conjugated primary antibody, resuspend cells in 350 μl of Incubation Buffer and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to Step 5.

Source / Purification

Background

Interleukin-1β (IL-1β), one of the major caspase-1 targets, is a multifunctional cytokine that is involved in a host of immune and proinflammatory responses (1). It is produced primarily by activated monocytes and macrophages. It signals through various adaptor proteins and kinases that lead to activation of numerous downstream targets (2-6). Human IL-1β is synthesized as a 31 kDa precursor. To gain activity, the precursor must be cleaved by caspase-1 between Asp116 and Ala117 to yield a 17 kDa mature form (7,8). Detection of the 17 kDa mature form of IL-1β is a good indicator of caspase-1 activity.